PROJECT SUMMARY Cardiovascular disease (CVD) due to atherosclerosis is the leading cause of mortality and disability globally. Even in patients treated with optimized standard-of-care regimens, residual morbidity and mortality remain high. New strategies that directly target atherosclerosis?the main cause of CVD?are urgently needed. Although recent conceptual advances have emphasized the importance of chronic inflammation and immune system activation in atherosclerosis in CVD, new promising anti-inflammatory agents tested in randomized clinical trials showed no benefits in either reducing cardiovascular end points or atherosclerotic plaque burden. The immune system is composed of a hierarchically organized set of molecular and cellular networks that act in concert in tissues and systemically. Yet, most studies of the role of immune system in atherosclerosis in humans have focused on circulating cells?with little or no focus on cellular immune infiltrates at the tissue site or the relationship between them. To gain a better understanding of systemic and local immune networks that contribute to CVD, we will take advantage of technological advances in molecular systems biology. Using CyTOF mass cytometry?a cutting-edge technique for high-dimensional analysis of immune cells?we identified single-cell variation and molecular activation of circulating peripheral blood mononuclear cells (PBMCs) and atherosclerotic tissue-associated immune cells in patients with carotid artery disease undergoing surgical revascularization. We will use a multi-tissue, systems biology approach to infer arterial wall- and blood-specific immune networks that govern the immune response at different stages of atherosclerosis. We will combine the use of innovative technologies such as CyTOF and RNA next-generation (NGS) to identify immune regulatory network relevant to human CVD disease. In specific Aim 1, we will identify the single-cell composition and molecular state of circulating and tissue-associated human immune cells. We will also determine the molecular and cellular correlates between systemic and tissue immune cell diversity and atherosclerotic and clinical phenotypes. In Specific Aim 2, we will identify immune networks in atherosclerosis and the clinical correlates of identified immune networks activated in atherosclerosis. The use of systems biology studies that integrate cutting-edge technologies to measure immune cell variation in patient samples to infer immune regulatory networks will deepen our knowledge of the contribution of the immune system to CVD and will set the stage to the selection of molecular targets for novel anti-inflammatory therapies.